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Effect of Alzheimer's brain extracts on dynein immunoreactivity in PC12 cells
1Division of Life Sciences, University of Texas at San Antonio, 78249, USA.
Abstract:
The neurodegenerative process in Alzheimer's disease (AD) has been suggested to occur as a consequence of microtubule disruption and subsequent loss of intracellular transport. Structural microtubule-associated proteins (MAPs) have been investigated for their role in the etiology of AD, but dynein, a force-producing MAP which mediates intracellular transport, has not been examined. In this report, dynein (MAP1C) immunoreactivity in AD brain tissue homogenates was observed increased 3.7-fold compared with control brain homogenate preparations. Similarly, NGF-differentiated PC12 cells cultured in the presence of soluble extracts prepared from AD brain tissue homogenates, exhibited an approximate 15-fold increase in dynein immunoreactivity compared to that of control brain tissue extracts. In contrast, AD clarified extracts had little effect upon "kinesin-like" protein immunoreactivity increased (approximately 2-fold); whereas, tau immunoreactivity was observed to be moderately increased (5-fold) over that of control brain extract treated PC12 cells. Chemical dephosphorylation and alkaline phosphatase treatment of AD extract-treated PC12 cell lysate prior to Western blotting resulted in complete loss of immunoreactivity, suggesting the dynein being monitored is a phosphorylated isoform. Furthermore, treatment of clarified brain tissue extracts with trypsin and (NH4)2SO4 suggests the endogenous elements giving rise to increased PC12 cell dynein intermediate chain immunoreactivity to be proteinaceous in nature. The observed increase in dynein intermediate-chain dynein immunoreactivity following exposure of neuronal cells to endogenous elements of AD brain may be reflective of dynein-microtubular array differences. Such an approach may be useful in assessing the effect of endogenous biomolecules on retrograde axonal transport in neuronal culture models.
Insights
Alzheimer's disease brain tissue shows increased dynein (MAP1C) levels, a motor protein crucial for intracellular transport. This suggests a potential role for dynein dysfunction in Alzheimer's neurodegeneration.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Alzheimer's disease (AD) involves neurodegeneration linked to microtubule disruption and impaired intracellular transport.
- Microtubule-associated proteins (MAPs) are implicated, but the role of dynein, a force-producing MAP, remains unexplored.
Purpose of the Study:
- To investigate dynein (MAP1C) immunoreactivity in Alzheimer's disease (AD) brain tissue and its response in neuronal cell models.
- To explore the nature of endogenous factors in AD brain extracts that influence dynein levels.
Main Methods:
- Quantification of dynein (MAP1C) immunoreactivity in AD and control human brain homogenates.
- Exposure of NGF-differentiated PC12 cells to AD and control brain extracts, followed by Western blotting for dynein, kinesin-like proteins, and tau.
- Biochemical treatments including dephosphorylation, alkaline phosphatase, trypsin, and ammonium sulfate precipitation.
Main Results:
- Dynein (MAP1C) immunoreactivity was significantly elevated (3.7-fold) in AD brain homogenates.
- PC12 cells treated with AD brain extracts showed a 15-fold increase in dynein immunoreactivity, while kinesin-like proteins increased ~2-fold and tau ~5-fold.
- The increased dynein was identified as a phosphorylated isoform, and the responsible factors in AD extracts were proteinaceous.
Conclusions:
- Elevated and phosphorylated dynein in AD brain tissue suggests its potential involvement in the disease's neurodegenerative processes.
- Endogenous AD brain components can modulate dynein levels in neuronal cells, potentially impacting retrograde axonal transport.
- This study highlights a novel approach to assess biomolecular effects on neuronal transport in AD models.